Survival motor neuron (SMN) gene: a Gene for spinal muscular atrophy

ABSTRACT

The present invention relates to the discovery of the human survival motor-neuron gene or SMD gene, which is a chromosome 5-SMA (Spinal Muscular Atrophy) determining gene. The present invention further relates to the nucleotide sequence encoding the SMN gene and corresponding amino acid sequence, a vector containing the gene encoding the SMN protein or a DNA sequence corresponding to the gene and transformant strains containing the SMN gene or a DNA sequence corresponding to the gene.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the discovery of the human survivalmotor-neuron gene or SMN gene which is a chromosome 5-SMA (SpinalMuscular Atrophy) determining gene. The present invention furtherrelates to the nucleotide sequence encoding the SMN gene andcorresponding amino acid sequence, a vector containing the gene encodingthe SMN protein or a DNA sequence corresponding to the gene andtransformant strains containing the SMN gene or a DNA sequencecorresponding to the gene.

More particularly, the present invention relates to means and methodsfor detecting motor neuron diseases having symptoms of muscular weaknesswith or without sensory changes such as amytrophic lateral sclerosis(ALS), spinal muscular atrophy (SMA), primary lateral sclerosis (PLS),arthrogryposis multiplex congenita (AMC), and the like. The methods fordetecting such motor neuron diseases include, but are not limited to,the use of specific DNA primers in the PCR technique, the use ofhybridization probes and the use of polyclonal and monoclonalantibodies.

Even more particularly, the present invention relates to the use of thehuman SMN gene or part of the gene, cDNA, oligonucleotide or the encodedprotein or part thereof in therapy by insertion of the human SMN gene orpart of the gene, cDNA, oligonucleotide or the encoded protein or partthereof, if required, into engineered viruses or vectors that serve asharmless carriers to transport the gene or part of the gene, cDNA,oligonucleotide or the encoded protein or part thereof to the body'scells including bone marrow cells.

The invention further relates to antigen sequences directed to the SMNgene.

In order to provide means for the therapy of motor neuron diseases, theinvention also relates to the protein encoded by the SMN gene.

The present invention also relates to the isolation of the mouse SMNgene, the nucleotide sequence encoding the mouse SMN gene andcorresponding amino acid sequence. A transgenic mouse model thathyperexpresses all or part of the SMN gene and a transgenic mouse modelproduced by homologous recombination with a mutated SMN gene is alsodescribed.

2. State of the Art

Degenerative motor neuron diseases can be placed into three majorcategories. Amyotrophic lateral sclerosis or ALS, motor neuron diseasessuch as spinal muscular atrophy (SMA) and motor neuron diseasesassociated with other degenerative disorders such as primary lateralsclerosis (PLS).

Amyotrophic lateral sclerosis (ALS) is the most frequently encounteredform of progressive neuron disease and is characteristically a disorderof middle age. The disease is characterized by progressive loss of motorneurons, both in the cerebral cortex and in the anterior horns of thespinal cord, together with their homologues in some motor nuclei of thebrainstem. It typically affects both upper and lower motor neurons,although variants may predominantly involve only particularly subsets ofmotor neurons, particularly early in the course of illness.

ALS is evidenced by the development of asymmetric weakness, with fatigueand cramping of affected muscles. The weakness is accompanied by visiblewasting and atrophy of the muscles evolves and over time, more and moremuscles become involved until the disorder takes on a symmetricdistribution in all regions, including muscles of chewing, swallowingand movement of the face and tongue. Fifty percent of patients havingALS can be expected to die within three to five years from the onset ofthe disease. Presently, there is no treatment that has influence on thepathologic process of ALS.

Spinal muscular atrophies (SMA) are characterized by degeneration ofanterior horn cells of the spinal cord leading to progressivesymmetrical limb and trunk paralysis associated with muscular atrophy.SMA represents the second most common fatal, autosomal recessivedisorder after cystic fibrosis (1 out 6000 newborns). Childhood SMA isclassically subdivided into three clinical groups on the basis of age ofonset and clinical course. The acute form of Werdnig-Hoffmann disease(Type I) is characterized by severe generalized muscle weakness andhypotonia at birth or in the 3 months following birth. Death, fromrespiratory failure, usually occurs within the first two years. Thisdisease may be distinguished from the intermediate (Type II) andjuvenile (Type III, Kugelberg-Welander disease) forms. Type II childrenwere able to sit but unable to stand or walk unaided, and they livebeyond 4 years. Type III patients had proximal muscle weakness, startingafter the age of two. The underlying biochemical defect remains unknown.In addition there is known to exist a slowly evolving adult form of SMA,sometimes referred to as SMA IV.

Primary lateral sicerosis (PLS) is a variant of ALS and occurs as asporadic disease of late life. Neuropathologically in PLS there is adegeneration of the corticospinal (pyramidal) tracts, which appearalmost normal at brainstem levels but become increasingly atrophic asthey descend through the spinal column. The lower limbs are affectedearliest and most severely.

Arthrogryposis Multiplex Congenita (AMC) is a frequent syndromecharacterized by congenital joint fixation (incidence of 1 out of 3000live births) resulting from decreased fetal movements in utero (Stern,W. G., JAMA, 81:1507-1510 (1923) ; Hall, J. G., Clin. Orthop., 194:44-53(1985)). AMC has been ascribed to either oligo-hydramnios or a varietyof diseases involving the central nervous system, skeletal muscle, orspinal cord. Since neuronal degeneration and neuronophagia occur in theanterior horns, it has been hypothesized that the AMC of neurogenicorigin could be related to acute spinal muscular atrophy; SMA Type IWerdnig-Hoffman disease (Banker, B. Q., Hum. Pathol., (1986);117:656-672).

The detection and clinical diagnosis for ALS, AMC, SMA and PLS is quitelimited to muscle biopsies, the clinical diagnosis by a physician andelectromyography (EMG). For example, the clinical criteria fordiagnosing SMA is set forth in the Clinical Criteria International SMAConsortium (Munsat T. L., Neuromuscular Disorders, Vol. 1, p. 81(1991)). But due to the complications of the various tests to detectmotor neuron disorders, the clinician usually attempts to eliminatevarious categories of other disease states such as structural lesions,infections, intoxications, metabolic disorders and heriditarybiochemical disorders prior to utilizing the above-described testmethods.

Presently there is no treatment for any of the above-mentioned motorneuron disorders. Basic rehabilitative measures, including mechanicalaids of various kinds, may help patients that have these diseasesovercome the effects of their disabilities, but often confiningrespiratory support systems are necessary to have the patient survivelonger.

Accordingly, it is an object of the present invention to characterizethe SMA gene which is responsible for SMA disorders and to clone the SMAgene into a vector, for example a plasmid, a cosmid, a phage, a YACvector, that can be used in the transformation process to produce largequantities of the SMN gene and SMN protein.

In yet another aspect of the invention is the use of primers andhybridization probes to detect and diagnose patients having motor neurondisorders such as AMC, ALS, SMA and PLS. Yet another aspect of thepresent invention is the use of the SMN gene or part thereof or cDNA,oligonucleotides, protein or part thereof in therapy to correctdisorders present in, for example AMC, SMA, ALS and PLS patients,especially gene disorders.

In yet another aspect, the present invention provides monoclonal andpolyclonal antibodies for detection of SMN gene defects in SMA patients.

Another object of the present invention provides the characterization ofthe SMA gene in the mouse. A transgenic mouse model is presented thathyperexpresses all or part of the SMN gene or a transgenic mouse that byhomologous recombination with a mutated mouse SMN gene producesabnormalities in the SMN gene is also described.

According to a further aspect of the invention, the therapy of motorneuron diseases can involve the protein encoded by the SMN gene.

These and other objects are achieved by the present invention asevidenced by the summary of the invention, the description of thepreferred embodiments and the claims.

OBJECTS AND SUMMARY OF THE INVENTION

It is an object of the present invention to provide a novel humanSurvival Motor Neuron gene or SMN gene, its DNA sequence and amino acidsequence.

Another aspect of the present invention provides a novel mouse SurvivalMotor Neuron gene or SMN gene, its DNA sequence and amino acid sequence.

Yet another aspect of the present invention is the provision of a vectorwhich is capable of replicating in a host microorganism to provide largequantities of the human or mouse SMN protein.

Yet another aspect of the present invention is the provision of specificDNA sequences that can be used to detect and diagnose spinal muscularatrophy and other motor neuron disorders. These DNA sequences can beused as primers in the polymerase chain reaction to amplify and detectthe SMN gene sequence, a truncated or mutated version of the SMN genesequence or lack of said sequence which leads to the diagnosis of SMA,AMC, and other motor neuron disorders.

Yet another aspect of the present invention provides a transgenic mousethat hyperexpresses all or part of the SMN gene or a transgenic mousethat by homologous recombination with a mutated mouse SMN gene producesabnormalities in the SMN gene is also described.

The inventors have identified two genes respectively designated T-BCD541and C-BCD541, which are involved in motor neuron disorders.

The T-BCD541 gene is responsible for the motor neuron diseases of theSMA type, since its alteration either by partial or total deletion, bymutation or any other modification, is sufficient to lead to apathological state at the clinical electromyographic or musclemorphological levels.

The C-BCD541 gene is different from the T-BCD541 gene at the level ofthe cDNA, since two nucleotides are modified. This C-BCD541 gene isnevertheless not correctly processed during the transcription incontrols and patients suffering from motor neuron diseases. The genomicDNA of the C-BCD541 gene is not correctly spliced during thetranscription providing thus for an abnormal transcript. The differencebetween the splicing of the T-BCD541 and the C-BCD541 gene results fromdifferences in the sequence of the introns of these genes.

The present invention thus further characterizes the structure andorganization of the human SMN gene which was found to be approximately20 kb in length and consists of 9 exons interrupted by 8 introns. Thenucleotide sequence, amino acid sequence as well as the exon-intronboundaries of the human SMN gene is set forth in FIG. 10. Allexon-intron boundaries display the consensus sequence found in otherhuman genes. A polyadenylation consensus site is localized about 550 bpdownstream from the stop codon (FIG. 10). The entire intron/exonstructure of the SMN gene permits the characterizations of the SMN genemutations in SMA disease or other motor neuron diseases.

The present invention also defines means for the detection of genomicabnormalities relating to motor neuron diseases at the level of theT-BCD541 gene or at the level of the C-BCD541 gene.

The genes of the invention can be further defined in that each of themcomprise intronic sequences corresponding to the following sequences:

In the T-BCD541 gene

for intron n° 6: 5′ AATTTTTAAATTTTTTGTAGAGACAGGGTCTCATTATGTTGCCCAGGGTGGTGTCAAGCTCCAGGTCTCAAGTGATCCCCCTACCTCCGCCTCCCAAAGTTGTGGGATTGTAGGCATGAGCCACTGCAAGAAAACCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAAAGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATGTCTATATAGCTATTTTTTTTAACTTCCTTTTATTTTC CTTACAG 3′

for intron n° 7: 5′ GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACAGTTAAAAAGTTCAGATGTTAAAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTATGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCAT TTGCAG 3′

In the C-BCD541 gene;

for intron n° 6: AATTTTTAAATTTTTTGTAGAGACAGGGTCTCATTATGTTGCCCAGGGTGGTGTCAAGCTCCAGGTCTCAAGTGATCCCCCTACCTCCGCCTCCCAAAGTTGTGGGATTGTAGGCATGAGCCACTGCAAGAAAACCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAAAGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTTATTTTCCTTACAG*

for intron n° 7:                         *GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACAGTTAAAAAGTTCAGATGTTAGAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGT TCTAATTTCTCATTTGCAG*

In a preferred embodiment of the invention, the gene of the invention iscapable of hybridizing in stringent conditions with the sequence of FIG.3 used as probe.

As hereabove written, the invention further relates to a variant of theSMN gene, which variant is a C-BCD541 gene having a cDNA sequencecorresponding to the sequence of FIG. 2.

The invention also relates to cDNA sequences such as obtained from oneof the above genes. Such cDNA sequences are disclosed in FIGS. 2 and 3.Both of these cDNA sequence are capable of encoding a protein comprisingthe amino acid sequence described on FIG. 1.

Despite this capacity to encode for such a protein, the inventors havenoted that the C-BCD541 gene is able to produce in vivo this protein oris not able to produce it in a sufficient quantity due to the abnormalsplicing of the gene during the transcription. Thus, the presence of theC-BCD541 gene does not enable to correct in vivo the deficiency(deletion, mutation, . . . ) of the T-BCD541 gene responsible for themotor neuron diseases of the SMA type or other motor neuron disorders.

In a particular embodiment, the invention relates also to a nucleotidesequence comprising nucleotides 34 to 915 of the sequence of FIG. 3, orto a sequence comprising nucleotides 34 to 915 of the sequence of FIG.2.

These nucleotide sequences correspond to the coding sequence ofrespectively the T-BCD541 gene and C-BCD541 gene.

The introns of the hereabove described genes are also included in theapplication. Especially introns 6 and 7 have respectively the followingsequences:

For the T-BCD541 gene:

Intron 6: 5′ AATTTTTAAATTTTTTGTAGAGACAGGGTCTCATTATGTTGCCCAGGGTGGTGTCAAGCTCCAGGTCTCAAGTGATCCCCCTACCTCCGCCTCCCAAAGTTGTGGGATTGTAGGCATGAGCCACTGCAAGAAAACCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAAAGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATGTCTATATAGCTATTTTTTTTAACTTCCTTTTATTTTC CTTACAG 3′

Intron 7: 5′ GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACAGTTAAAAAGTTCAGATGTTAAAAAGTTGAAAGGTTAATGTAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTATGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAG 3′

For the C-BCD541 gene:

Intron 6: AATTTTTAAATTTTTTGTAGAGACAGGGTCTCATTATGTTGCCCAGGGTGGTGTCAAGCTCCAGGTCTCAAGTGATCCCCCTACCTCCGCCTCCCAAAGTTGTGGGATTGTAGGCATGAGCCACTGCAAGAAAACCTTAACTGCAGCCTAATAATTGTTTTCTTTGGGATAACTTTTAAAGTACATTAAAAGACTATCAACTTAATTTCTGATCATATTTTGTTGAATAAAATAAGTAAAATGTCTTGTGAACAAAATGCTTTTTAACATCCATATAAAGCTATCTATATATAGCTATCTATATCTATATAGCTATTTTTTTTAACTTCCTTTTATTTTCCTTACAG*

Intron 7:                         GTAAGTCTGCCAGCATTATGAAAGTGAATCTTACTTTTGTAAAACTTTATGGTTTGTGGAAAACAAATGTTTTTGAACAGTTAAAAAGTTCAGATGTTAGAAAGTTGAAAGGTTAATGAAAACAATCAATATTAAAGAATTTTGATGCCAAAACTATTAGATAAAAGGTTAATCTACATCCCTACTAGAATTCTCATACTTAACTGGTTGGTTGTGTGGAAGAAACATACTTTCACAATAAAGAGCTTTAGGATATGATGCCATTTTATATCACTAGTAGGCAGACCAGCAGACTTTTTTTTATTGTGATATGGGATAACCTAGGCATACTGCACTGTACACTCTGACATATGAAGTGCTCTAGTCAAGTTTAACTGGTGTCCACAGAGGACATGGTTTAACTGGAATTCGTCAAGCCTCTGGTTCTAATTTCTCATTTGCAG*

The invention further encompasses a nucleotide sequence, characterizedin that it comprises at least around 9 nucleotides and in that it iscomprised within a sequence which has been described above or in that ithybridizes with a sequence as described above in hybridizationconditions which are determined after choosing the oligonucleotide.

For the determination of the hybridization conditions, reference is madeto the hybridization techniques for oligonucleotides probes such asdisclosed in Sambrook et al. Molecular Cloning a Laboratory Manual 2ndedition, 1989.

The sequences of the invention are either DNA (especially genomic DNA orcDNA or synthetic DNA) or RNA. They can be used as probes for thedetection of the T-BCD541 or C-BCD541 genes or as primers for theamplification of genomic DNA present in a biological sample.

Preferred primers are those comprising or relating to the followingsequences: a) 5′ AGACTATCAACTTAATTTCTGATCA 3′ (R 111) b)5′ TAAGGAATGTGAGCACCTTCCTTC 3′ (541C770)

The above primers are characteristic of exon 7 of the T-BCD541 gene. (c)GTAATAACCAAATGCAATGTGAA (541C960) (d) CTACAACACCCTTCTCACAG (541C1120)

The above primers are characteristic of exon 8 of the T-BCD541 gene.

The primers used by pairs can form sets for the amplification of genomicDNA in order to detect motor neuron diseases.

Inverted complementary sequences with respect to the above primers canalso be used.

Preferred sets of primers are the following:

a pair of primers contained in the sequence comprising nucleotides 921to 1469 of the sequence of FIG. 3 and/or

a pair of primers comprising the following sequences:5′ AGACTATCAACTTAATTTCTGATCA 3  5′ TAAGGAATGTGAGCACCTTCCTTC 3′

Another preferred set of primers comprises:

a pair of primers having the following sequences:5′ AGACTATCAACTTAATTTCTGATCA 3′ 5′ TAAGGAATGTGAGCACCTTCCTTC 3′

a pair of primers having the following sequences:5′ GTAATAACCAAATGCAATGTGAA 3′ and/or 5′ CTACAACACCCTTCTCACAG 3′

From a general point of view for the detection of divergence in exon 7.between the T-BCD541 and C-BCD541 genes oligonucleotide primers can beselected in the fragment 5′ from the divergence and within exon 7 orintron 7.

Other primers that can be used for SSCP analysis for diagnostic purposesare selected from amongst the following: 5′EXON 1 121md/121me   Size:170bp 121MD    5′ AGG GCG AGG CTC TGT CTC A 121ME    5′ CGG GAG GAC CGC TTGTAG T EXON1 121ma/121mf   Size:180 bp 121MA    5′ GCC GGA AGT CGT CACTCT T 121MF    5′ GGG TGC TGA GAG CGC TAA TA EXON2A ex2A5/EX2A3  Size:242 bp EX2A5    5′ TGT GTG GAT TAA GAT GAC TC EX2A3    5′ CAC TTTATC GTA TGT TAT C EXON2B Ex2B5/EX23   Size:215 bp EX2B5    5′ CTG TGCACC ACC CTG TAA CAT G EX23    5′ AAG GAC TAA TGA GAC ATC C EXON3SM8C/161CR2   Size:238 bp SM8C    5′ CGA GAT GAT AGT TTG CCC TC 161CR2   5′ AG CTA CTT CAC AGA TTG GGG AAA G SM8D/C260   Size:150 bp SM8D   5′ CTC ATC TAG TCT CTG CTT CC 541C260    5′ TGG ATA TGG AAA TAG AGAGGG AGC EXON4 SM3CA/C460   Sizo:150 bp SM3CA    5′ CAC CCT TAT AAC AAAAAC CTG C 541C460    5′ GAG AAA GGA GTT CCA TGG AGC AG SM3CB/C380  Size:180 bp SM3CB    5′ GAG AGG TTA AAT GTC CCG AC 541C380    5′ GTGAGA ACT CCA GGT CTC CTG G EXON5 EX55/C590   Size:254 bp EX55    5′ TGAGTC TGT TTG ACT TCA GG 541C590    5′ GAA GGA AAT GGA GGC AGC CAG CEX53/C550   Size:168 bp EX53    5′ TTT CTA CCC ATT AGA ATC TGG 541C550   5′ CCC CAC TTA CTA TCA TGC TGG CTG EXON6 164C25/C849   Size:143 bp164C25    5′ CCA GAC TTT ACT TTT TGT TTA CTG 541C849    5′ ATA GCC ACTCAT GTA CCA TGA EX63/C618   Size:248 bp EX63    5′ AAG AGT AAT TTA AGCCTC AGA CAG 541C618    5′ CTC CCA TAT GTC CAG ATT CTC TTG 3′ EXON7R111/C770   Size:200 bp R111    5′ AGA CTA TCA ACT TAA TTT CTG ATC A541C770    5′ TAA GGA ATG TGA GCA CCT TCC TTC R111/C261   Size:244 bpR111    5′ AGA CTA TCA ACT TAA TTT CTG ATC A 164C261    5′ GTA AGA TTCACT TTC ATA ATG CTG INTRON7 164C45/164C265 Size:220 bp 164c45    5′ CTTTAT GGT TTG TGG AAA ACA 3′ 164c265    5′ GGC ATC ATA TCC TAA AGC TCEXON8 c960/C1120   Size:186 bp 541c960    5′GTA ATA ACC AAA TGC AAT GTGAA 543C1120    5′CTA CAA CAC CCT TCT CAC AG 164C140/C920 164C140   5′ GGT GTC CAC AGA GGA CAT GG 541C920    5′ AAG AGT TAA CCC ATT CCAGCT TCC

The invention also concerns antisense DNA or RNA, capable of hybridizingwith the C-BCD541 gene and particularly to the intron sequences,especially with the fragment of the introns which differ from thecorresponding part in the T-BCD541 gene.

The invention also relates to a protein comprising the amino acidsequence of FIG. 1, or to a protein having the amino acid sequence ofFIG. 8.

The protein relating to the sequence of FIG. 1 can be used in acomposition for the treatment of motor neuron diseases, via oral,intra-muscular, intravenous administration, or via administration in thespinal cord fluid.

The invention further provides a kit for the in vitro diagnosis of motorneuron diseases, comprising:

a set of primers as described above;

reagents for an amplification reaction and

a probe for the detection of the amplified product.

According to another embodiment of the invention, a kit for thedetection of the motor neuron diseases containing a hybridization probeas described above is provided.

Oligonucleotide probes corresponding to the divergences between thegenes can be used.

The diagnosis can be especially directed to SMA motor neuron pathology.

The invention also concerns cloning or expression vectors comprising anucleotide sequence as defined above. Such vectors can be, for example,plasmids, cosmids, phages, YAC, pYAC, and the like. Preferably, such avector has a motor neuron tropism. Especially for the purpose ofdefining means for gene therapy, it can be chosen among poliovirusvector, herpes virus, adenovirus, retrovirus vectors, synthetic vectorsand the like.

Within the scope of the invention are contemplated further recombinantsequences. The invention also concerns recombinant host cells, i.e.,yeasts, CHO cells, baculovirus, bone marrow cells, E. coli,fibroblasts-epithelial cells, transformed by the above recombinantsequences.

The invention also relates to a method for detecting motor neurondisorders including spinal muscular atrophy, amyo trophoc lateralsclerosis and primary lateral sclerosis, said method comprising thesteps of:

(a) extracting DNA from a patient sample;

(b) amplifying said DNA with primers as described above;

(c) subjecting said amplified DNA to SCCP;

(d) autoradiographing the gels; and

(e) detecting the presence or absence of the motor neuron disorder.

Steps (c) and (d) can be replaced by a step of digestion with BsrIenzyme or with any other enzyme capable of recognizing specifically thedivergence of the genes or mismatches in genes, or by sequencing.

The invention also relates to a method for detecting spinal muscularatrophy, said method comprising the steps of:

(a) extracting DNA from a patient sample;

(b) hybridizing said DNA with a DNA probe comprising all or part of thecDNA sequence of FIG. 3 or of FIG. 2 under stringent conditions; and

(c) detecting the hybrids possibly formed.

The invention also relates to a method for detecting arthrogryposismultiplex congenital said method comprising the steps of:

(a) extracting DNA from a patient sample;

(b) amplifying said DNA via PCR using unlabeled primers from exon 7 andexon 8 of the SMN gene;

(c) subjecting said amplified DNA to SCCP;

(d) autoradiographing the gels; and

(e) detecting the presence or absence of arthrogryposis multiplexcongenita.

Yet another method to detect arthrogryposis multiplex congenita concernsdinucleotide Repeat Polymorphism Analysis using genotyping markers C272and C212 after PCR amplification.

The present invention further concerns polyclonal antiserum ormonoclonal antibodies directed to the protein of FIG. 1, the protein ofFIG. 8 or the protein of FIG. 12.

Yet another aspect of the present invention is directed to the use ofthe entire or partial nucleotide sequence of SMN as a probe to detectSMA as well as to indetify and clone genes related to SMN gene motorneuron in animals or organisms.

Yet another aspect of the present invention is the use of the SMAprotein to produce polyclonal and monoclonal antibodies, whichantibodies may be used to detect and diagnose SMA.

In another aspect, polyclonal rabbit antiserum were generated againstsynthetic peptides corresponding to the amino acid sequence of FIGS. 1,8 and 12, including the amino acid terminus and the carboxy terminus.

Accordingly, in one of its process aspects, the present inventionrelates to the detection of SMA in patients having SMA or related motorneuron disorders such as AMC, ALS and PLS.

Yet another aspect of the present invention is to administer the SMNgene part thereof, cDNA or oligonucleotides to patients who are eitherlacking the gene or have a genetically defective gene as such or afterincorporation into engineered viruses or vectors.

These and other aspects of the present invention will be discussed indetail below in the preferred embodiments of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the amino acid sequence of the SMN coding region of the cloneT-BCD541.

FIG. 2 is the nucleotide sequence of the SMN coding region as well asthe 5′ and 3′ flanking regions of clone C-BCD541; the coding region isunderlined.

FIG. 2B contains the sequence starting from intron 6 up to exon 8 of theC-BCD541 gene. The underlined sequences are those of exons 7 and 8.Sequences of introns 6 and 7 can be chosen as oligonucleotides toamplify the cDNA region allowing the distinction, within exon 7, betweenthe T-BCD541 gene and the C-BCD541 gene. The position of the divergentnucleotides between the T-BCD541 and C-BCD541 cDNA are in italics.

FIG. 3A is the nucleotide sequence of the SMN coding region as well asthe 5′ and 3′ flanking regions of clone T-BCD541. The coding sequencesare underlined. The numbers of the exons are indicated on the sequence.Asteriks indicate the beginning of each exon. The nucleotides which areindicated in italics are those which differ between the C-BCD541 and theT-BCD541 genes.

FIG. 3B represents the sequence from intron 6 up to the end of exon 8 ofthe T-BCD541 gene. The sequence of exons 7 and 8 is underlined.

FIG. 4 is the nucleotide sequences of the markers C212, C272, C171,AFM157xd10, and C161.

FIG. 5 represents various probes utilized in the present inventionrevealing several loci that the probes hybridized to.

FIG. 6 represents the telomeric element containing the survival SMNgene.

FIG. 7 represents the marked decrease of gene dosage with probe 132SEII,mapping close to this.

FIG. 8 represents the amino acid sequence of the truncated SMN protein.

FIG. 9 is a schematic representation of the genomic structure of thehuman SMN gene. The designations and positions of genomic clones areshown above the figure. L-132, L-5, and L-13 depict the genomic clonesspanning the entire SMN gene, while L-51 spans part of exon 1. Microsatellites and DNA markers are indicated above the genomic map. B, H,and E mean BgIII, HindIII and EcoRI, respectively. C212, p322, C272,132SEII and C171 represent various markers. 1, 2a, 2b, 3, 4, 5, 6, 7,and 8 represent exons of the SMN and C-BCD541 genes. The entire sequenceof L-132 is obtained by PCR amplification from exon 1 to exon 2A.

FIG. 10 represents the nucleotide sequence and amino acid sequence ofthe entire human SMN gene including the introns and exons. Translatednucleotide sequences are in upper case, with the corresponding aminoacids shown below that. The polyadenylation signal is in bold face.Arrowheads indicate the position of the single base differences betweenSMN and C-BCD541 genes in introns 6 and 7 and exons 7 and 8. Italicletters indicate the position of the oligonulcoeitdes chosen for thedetection of divergences in intron 7. (*) indicates the position of thestop codon.

FIG. 11 represents the nucleotide sequence upstream of the coding regionof the human SMN gene and illustrates the presence of putative bindingsites for the transcription factors of AP-2, GH-CSE2, DTF-1, E4FI,HINF-A, H4TF-1, β-IFN and SpI. Bold letters indicate the dinucleotiderepeat (CA) corresponding to the C272 markers.

FIG. 12 represents the nucleotide and amino acid sequences of Mouse SMNcDNA. (*) indicates the position of the stop codon.

FIG. 13 represents a comparative analysis of the amino acid sequence ofhuman SMN (above) and mouse SMN (below).

FIG. 14 illustrates the genetic analysis of family 6 Lane A showsevidence of inherited maternal deletion seen with the microsatellitemarker C272 as the proband inherited only allele from the father. LanesB and C represent SSCP analysis of PCR-amplified exons 7 (lane B) and 8(lane C) of SMN (closed arrowheads) and its centromeric copy (openarrowheads). “F” represents the father, “M” the mother, and “A” theaffected infant.

FIG. 15 illustrates the band shifts on single strand confirmationpolymorphism (SSCP) analysis of the PCR amplified intron 7 and permittedindetification of SMN (closed arrowheads) and its centromericcounterpart C-BCD541 (open arrowheads).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

As used herein, the term “contig” means overlapping nucleotidesequences.

Previous studies by means of linkages analysis have shown that all threeforms of spinal muscular atrophy map to chromosome 5q11.2-q13.3. (L. M.Brzustowicz et al. Nature, 344, 540 (1990); J. Melki et al, Nature, 345,823 (1990); J. Melki et al, Lancet, 336, 271 (1990). A yeast artificialchromosome (YAC) contig of the 5q13 region spanning the disease locuswas constructed that showed the presence of low copy-repeats in thisregion. Allele segregation was analyzed at the closest genetic locidetected by markers derived from the YAC contig (C212. C272 and C161) in201 SMA families. These markers revealed two loci (C212, C272) or threeloci on the 5q13 region (C161). Inherited and de novo deletions wereobserved in 9 unrelated SMA patients. Moreover, deletions were stronglysuggested in at least 18% of SMA type I patients by the observation ofmarked heterozygosity deficiency for the loci studied. These resultsindicated that deletion events are statistically associated with thesevere form of SMA.

By studying all polymorphic DNA markers derived from the YAC contig, itwas observed that the smallest rearrangement occured within a regionbordered by loci detected by C161 and C212-C272 and entirely containedin a 1.2-Mb YAC clone 903D1. See, for example, French Patent ApplicationNo. 9406856 incorporated herein by reference.

The present invention characterized the small nested critical SMA regionof about 140 Kb by a combination of genetic and physical mapping in SMApatients. This region suggested a precise location for the SMA gene andtherefore, a limited region within which to search for candidate genes.The present invention identified a duplicated gene from the 5q13 region.One of them (the telomeric gene) is localized within the criticalregion. Moreover, this gene was lacking in 213 out of 230 (92.2%) orinterrupted in 13 out of 230 (5.6%) SMA patients. In patients where theteiomeric gene is not lacking or interrupted, deleterious mutationsindicated that this telomeric gene, termed survival motor-neuron (SMN)gene, is the chromosome 5 SMA-determining gene.

The SMN gene was discovered using a complex system of restrictionmapping, distinguishing the E^(Tel) from the E^(Cen) by Southern blot,and the determination of the differences between the E^(Tel) in SMApatients by genetic and physical mapping. After confirming the locationof the SMN gene, a phage contig spanning the critical region of thetelomeric element was constructed to identify specific clones containingthe SMN gene.

Analysis of the SMN gene in SMA patients compared with those of normalpatients revealed either the SMN gene was either lacking or truncated in98% of SMA patients or had combined mutations not present in normalcontrol patients.

To identify a large inverted duplication and a complex genomicorganisation of the 5q13 region, long-range restriction mapping usingpulsed field gel electrophoresis (PFGE) of the YAC contig was performed.

YACs were ordered by comparing their haplotypes with that of the humandonor at the polymorphic loci detected markers C212, C272. C171 and C161(FIG. 4).

The restriction enzymes SacII, BssHII, SfiI, EagI and XhoI were used todigest the YACs containing the telomeric loci detected by markers C212,C272, C171 and C161 (YAC clone 595C11), the centromeric loci detected bythese markers (YAC clones 121B8, 759A3, 278G7) or both (YAC clones 903D1and 920C9). Lambda phage libraries of YACs 595C11, 121B8 and 903D1 wereconstructed and subclones from phages containing markers C212 (p322),C272 (132SE11), C161(He3), AFM157xd10(131xb4) and CMS1 (p11M1) were usedas probes for PFGE analysis. FIG. 5 shows that probes 132SE11, 11P1 andp322 revealed two loci, and probe He3 revealed 4 loci on the YAC contig,whereas probe 131xb4 revealed several loci on 5p and 5q13. Therestriction map (FIG. 6) showed that the 5q13 region contained a largeinverted duplication of an element (E) of at least 500 Kb, termedE^(Tel) and E^(Cen) for the telomeric and centromeric elements,respectively.

The PFGE analysis of SMA and control individuals revealed a high degreeof variability of restriction fragments which hampered thedistinghishment of E^(Tel) from the E^(Cen) and the recognition ofabnormal restriction fragments in SMA patients.

In order to distinguish between the E^(Tel) and the E^(Cen), a Southernblot analysis was then performed. The Southern blot was performed by themethods described in Sambrook et al, supra.

More specifically, DNA from YAC clones, controls and SMA patients wasdigested with restriction enzymes SacI, KpnI, MspI, PstI, PvuII, EcoRI,HindIII, BgIII and XbaI for Southern blotting and hybridized with clones132SE11, 11p1, He3, 131xb4 and p322 as probes. None of the probes exceptone (He3) detected a difference between the two duplicated elements.Three HindIII restriction fragments of 12, 11 and 3.7 Kb were detectedby probe He3. A 12 Kb HindIII restriction fragment was detected in YACclones 754H5 and 759A3, indicating that this fragment corresponded tothe most centromeric locus in the E^(Cen). Conversely, a 11 Kb HindIIIfragment was detected in YACs clones 595C11, 903D1 and 920C9 indicatingthat this fragment corresponded to a single locus on the E^(Tel).Finally, a 3.7 Kb HindIII fragment was noted in non-overlapping YACscontaining either E^(Tel) or E^(Cen), indicating that this fragmentcorresponded to two different loci. Similar results were obtained withSacI and KpnI. The three restriction fragments detected by He3 wereobserved on the monochromosomal hybrid HHW105 (Carlock, L. R. et al, Am.J. of Human Genet., 1985, Vol. 37, p. 839) and in 30 unrelated, healthyindividuals, confirming that these fragments were not due topolymorphisms. The Southern analysis results allowed one to distinguishE^(Tel) from the E^(Cen) in both controls and SMA patients.

Thus, once the E^(Tel) from the E^(Cen) was distinguished, it wasnecessary to determine the differences between the E^(Tel) in SMApatients and those of the normal control. This was done by using geneticand physical mapping. This genetic and physical mapping identifiedgenomic rearrangements in the telomeric element of E^(Tel) of SMApatients.

It was previously shown that 9 out of 201 (9/201) SMA patients displayedlarge-scale deletions encompassing either one or the two loci detectedby markers C212 and C272 on one mutant chromosome (J. Melki et al,Science, 264, 1474 (1994)). On the other hand, 22 out of 30 (22/30)patients born to consanguineous parents including 13 out of 14 (13/14)type I and 9 out of 10 (9/10) type III SMA, were homozygous by descentfor the most closely flanking polymorphic markers.

The genomic DNA of the 9 patients harboring large scale deletions andthe 22 consanguineous patients displaying homozygosity by descent weredigested with HindIII for Southern blotting and hybridized with probeHe3. The 11 Kb fragment revealed by probe He3 was absent in 12 out of 13(12/13) consanguineous type I patients. In 2 out of 12 (2/12), thedeletion also involved the 3.7 Kb fragment. By contrast, the 11 Kbfragment was absent in 1 out of 8 (1/8) consanguineous type III patientsonly. Consistently, the 11 Kb HindIII fragment was absent in 4 out of 9(4/9) patients harboring large scale deletions on one mutant chromosome.Of particular interest was the absence of the 11 Kb fragment in thepatient harboring a deletion of one of the two loci detected by markersC212 and C272.

When analyzed together, these observations provided evidence for genomicrearrangements of E^(Tel) in SMA patients and supported the location ofthe SMA gene centromeric to the locus revealed by the 11 Kb HindIIIfragment, since all consanguineous type III patients but one were notdeleted for this locus.

In order to characterize the centromeric boundary of the genomicrearrangement in the disease, the allele segregation at loci detected bymarker C272 in consanguineous SMA patients was analyzed. Allconsanguineous SMA type I patients had one single PCR amplificationproduct, compared with 0 out of 60 controls. This marked heterozygositydeficiency was due to deletion of one of the two loci detected by C272,as indicated by the marked decrease of gene dosage with probe 132SE11.mapping close to this marker. By contrast, 7 out of 9 (7/9)consanguineous type III SMA patients had two C272 amplification productsinherited from both parents, indicating homozygosity at each locusdetected by marker C272. Moreover, no gene dosage effect was observedwith probe 132SE11 indicating the absence of deletion involving thelocus detected by C272 in type III consanguineous patients.

Assuming that the same locus is involved in all three types of SMA,these results indicate that the disease causing gene is distal to thetelomeric locus detected by C272.

These studies place the SMA gene within the telomeric element E^(Tel),between the telomeric loci detected by markers C272 and He3 (11 kbHindIII fragment). Based on long-range restriction mapping using PGFE ofthe YAC contig, this critical region is entirely contained in a 140 KbSacII fragment of YAC clone 903D1 (or 150 Kb SacII fragment of YAC clone920D9).

After confirming that the SMN gene was located on a 140 Kb SacIIfragment a phage contig spanning the critical region of the telomericelement was constructed in order to identify and characterize the SMNgene.

Phage clones containing markers C212, C272, C171 and C161 were isolatedfrom the λ phage libraries constructed from YAC clones 595C11 and 903D1and used as a starting point for bidirectional walking. A phage contig(60 Kb) surrounding markers C212, C272 and C171 was constructed based onthe restriction map of the phage clones (FIG. 6).

To identify genes in the contig, the following three stategies wereused:

1 ) a search for interspecies-conserved sequences was conducted;

2) exon trapping method was performed; and

3) direct cDNA selection was performed. The genomic probe 132SE11,derived from the phage containing the marker C272, gave positivehybridization signals with hamster DNA indicating the presence ofinterspecies-conserved sequences. The screening of a λgt10 human fetalbrain cDNA library with probe 132SE11 resulted in the selection of 7overlapping λ clones spanning 1.6 kbp. Sequence analysis of the clonesrevealed a 882 bp open-reading frame (ORF) and a 580 bp non-codingregion. A 1.5 kbp clone (BCD541) contained the entire coding sequenceand most of the 3′ non-coding region. The 3′ end of the cDNA along withits poly(A)⁺ tail was obtained by PCR-amplification of a lymphoblastoidcell line cDNA library.

Two cDNA clones lacked nucleotides 661 to 755, suggesting that analternative splicing might have occured. Northern blot analysis ofpoly(A)⁺ RNA from various tissues including heart, brain, liver, muscle,lung, kidney and pancreas, revealed the presence of a widely expressed1.7 kb transcript. The ORF encodes a putative protein of 294 amino acidswith a predicted molecular weight of approximately 32 Kd.

A homology search using the FASTA and BLAST networks failed to detectany homology at either the nucleotide or the amino acid level.

To further distinguish whether there was any duplication of the BCD541gene in the 5q13 region, BCD541 cDNA was used as a probe for Southernblot and PFGE analysis of YAC clones spanning the disease locus.

Specific hybridization with non-overlapping YACs containing either theE^(Cen) only (YAC clones 759A3, 121B8 and 278G7), or containing theE^(Tel) only (YAC clone 595C11) provided evidence for duplication of theBCD541 gene. Each gene encompassed approximately 20 kb and displayed anidentical restriction pattern. Evidence for head to head orientation ofthe two genes was derived from the location of the SacII and EagIrestriction sites of the non-overlapping YAC clones containing eitherE^(Cen) or E^(Tel), following hybridization experiments with probesBCD541 and p322 which flank the SacII and EagI sites of each element.

In order to look for divergences in the two copies of the BCD541 gene,the organization of the telomeric gene was characterized and compared tothat of the centromeric counterpart. Genomic sequence analysis revealedthat the telomeric BCD541 gene is composed of 8 exons (FIG. 3). However,it is now known that the previously known exon 2 is composed of 2 exonsseparated by an additional intron as set forth in FIG. 10, therefore theSMN gene is composed of 9 exons.

Starting from either the centromeric or telomeric gene loci (in YACclones 121B8 and 595C11, respectively), PCR-amplification and sequenceof each exon and their flanking regions revealed five discrepanciesbetween the centromeric and the telomeric BCD541 genes. The first one isa conservative substitution in exon 7 (codon 280) specific for thetelomeric (TTC) or the centromeric BCD541 gene (TTT). The second one,located in the 3′ non-coding region (exon 8 nucleotide n° 1155) isspecific for the telomeric (TGG) or the centromeric BCD541 gene (TGA).Three other single base substitutions were observed in the sixth andseventh introns.

The observation of both versions of each exon (exon 7 and 8) on eitherYAC clones containing both gene loci (YAC clone 920C9) or themonochromosomal hybrid HHW105 demonstrated that these substitutions areneither allelic nor due to polymorphisms. Band shifts on SSCP analysisof amplified exons 7 and 8 allowed an easy distinction of the telomeric(T-BCD541) and centromeric genes (C-BCD541) in both controls and SMApatients. All the unrelated healthy controls testeo (n=75) harbored theT-BCD541 gene as determined by SSCP analysis of exons 7 and 8 (100%).Most of them (89.3%) also harbored the C-BCD541 gene but 8 out of 75(8/75) (10.7%) lacked the C-BCD541.

A total of 230 SMA patients were tested for single base substitutionsdetected in exons 7 and 8 by SSCP method after PCR-amplification ofgenomic DNA. Among them, 103 belonged to type I, 91 to type II, and 36to type III. Interestingly, 213 out of 230 SMA patients (92.6%) lackedthe T-BCD541 gene on both mutant chromosomes (compared with 0 out of 75controls (0%). Moreover, 13 out of 230 SMA patients (5.6%) lacked theT-BCD541 gene for exon 7 on both mutant chromosomes but retained theT-BCD541 gene for exon 8 compared with 0 out of 75 controls (0%).Finally, only 4 out of 230 SMA patients (1.7%) harbored the T-BCD541gene as determined by SSCP analysis of exons 7 and 8.

These results show that the T-BCD541 gene is either lacking or truncatedin 98% of SMA patients. In addition, these data support the view thatthe disease gene is located between the telomeric locus detected by C272and exon 8 of the T-BCD541 gene. Therefore, according to the overlappingrestriction map of the phage contig, the critical region is entirelycontained in 20 kb, suggesting that the telomeric BCD541 gene is thechromosome 5 SMA-determining gene.

In order to demonstrate that the T-BCD541 gene is responsible for SMA,point mutations in the 4 SMA patients in whom no rearrangement of theT-BCD541 gene had been observed were searched. Direct sequencing of PCRamplification products of each exon with their flanking regions wasperformed in the four patients.

A 7 bp deletion in the 3′ splice acceptor site of intron 6(polypyrimidine tract) was found in patient SA. Sequence analysis ofexon 7 flanking the deleted intron, recognized the sequence specific forthe T-BCD541 gene. Moreover, the non-deleted PCR-product correspondingto the same region, harbored the sequence specific for the C-BCD541suggesting that the other mutant allele lacked the T-BCD541 gene.

In patient BI, a 4 bp deletion in the 5′ consensus splice donor site ofintron 7 was found. This deletion occured on the T-BCD541 gene asdetermined by sequence analysis of the flanking exon 7.

In patient HU, a point mutation in codon 272 (TAT→TGT) was found. Thismutation changed a Tyrosine to Cysteine. The patient was heterozygousfor the mutation, presumably carrying a different SMA mutation on theother allele. All three mutations observed in patients SA, HU and BIwere not detected in 100 normal chromosomes ruling out rarepolymorphisms.

A different splicing of exon 7 distinguished the C-BCD541 from theT-BCD541 gene using reverse transcription-based PCR. Eleven SMA patientswere selected for the analysis of their transcripts by Northern blot orreverse transcription-based PCR amplification. Eight of them belonged totype I, 1 to type II and 2 to type III. SSCP analysis of genomic DNAshowed an absence of T-BCD541 gene in 10 patients and one patient (SA)had C-BCD541 and T-BCD541 genes for both exons 7 and 8. Six unrelatedcontrols who harbored both C-BCD541 and T-BCD541 genes arid 2 controlswith only T-BCD541 gene were included in the present study.

The expression of this gene in lymphoblasts made it possible to analyzethe BCD541 transcripts in cell lines derived from controls and SMApatients. Northern blot analysis of RNA from lymphoblastoid cell linesshowed the presence of a 1.7 kb mRNA in all samples. None of the SMApatients showed a transcript of altered size. It was observed that areduced level of transcripts was obtained when compared to theexpression of the β-actine gene in 3 out of 4 type I SMA patients.Normal mRNA level were found for the other SMA probands.

Since the Northern blot analysis revealed the presence of a transcriptin SMA patients who had the C-BCD541 gene only for both exons 7 and 8 asdetermined by SSCP analysis, these results indicated that both C-BCD541and T-BCD541 genes were expressed. To prove whether both BCD541 geneswere expressed, RT-based PCR amplification of RNA isolated from thelymphoblastoid cell lines from controls and SMA patients was used.Direct sequencing of PCR products flanking exons 7 and 8 revealed thatpatients who had C-BCD541 only displayed the sequence specific for theC-BCD541 gene. Controls who had both T-BCD541 and C-BCD541 genes, hadtwo types of transcripts corresponding to both BCD541 genes. Theseresults confirmed that both genes were expressed. In addition, 2alternative splicings involving exon 5 or exon 7 that resulted indifferent transcripts were observed. The alternative splicing of exon 5confirmed previous sequence data on the cDNA clones.

The analysis of the RT-PCR amplification products encompassing exons 6to 8 showed that the spliced transcript keeping exon 7. was present incontrols who had both C-BCD541 and T-BCD541 genes or controls who hadthe T-BCD541 gene only. Conversely, the alternative spliced transcriptlacking exon 7 was observed in controls who had both genes, but not incontrols who had the T-BCD541 gene only. These results indicated thatthe alternative spliced transcript lacking exon 7 was derived from theC-BCD541 gene only.

The transcript analysis of patient SA harboring a 7 bp deletion of the3′ splice acceptor site of intron 6 of the T-BCD541 gene revealed thepresence of both spliced transcript keeping exon 7 and alternate splicedtranscript lacking exon 7. Moreover, the sequence analysis ofamplification products from the spliced transcript keeping exon 7,showed a sequence specific for the C-BCD541 gene (FIG. 2). These resultsdemonstrated that the 7 bp deletion of intron 6 observed in patient SAwas deleterious for the correct splicing of exon 7 of T-BCD541 geneonly. In addition, because a differential splicing of exon 7 allowed oneto distinguish the 2 BCD541 genes, this difference was analyzed amongcontrols and SMA patients including patient SA. In controls, the amountof alternated spliced transcript lacking exon 7 was less abundant thanthat of spliced product keeping exon 7. Conversely, in SMA patients, theamount of alternated spliced transcript lacking exon 7 was equal or moreabundant than that of spliced product keeping exon 7.

These results provide evidence for a difference between controls and SMApatients at the transcription level of these genes. The alternativespliced transcript lacking exon 7 resulted in a shorter ORF with adifferent C-terminus protein that might have effects on the proteinfunction.

To further characterize the entire structure and organization of thehuman SMN gene, three genomic clones were isolated from a FIX II phagelibrary derived from YAC clone 595C11 and screened with the full-lengthBCD541 cDNA (FIG. 2A) as a probe. After selecting several clones thathybridized to the probe, restriction mapping and Southern blot analysisindicated that phages L-132, L-5 and L-13 spanned the entire SMN gene.

These three phage clones were further subjected to sequencing using theMaxam-Gilbert or Sanger et al methods of sequencing disclosed inSambrook et al supra.

The nucleotide and amino acid sequence of the entire SMN gene includingexons and introns is set forth in FIG. 10. The human gene isapproximately 20 kb in length and consists of nine (9) exons interruptedby 8 introns as shown in FIG. 10. The human SMN gene has a molecularweight of approximately 32 kDA.

Although it was thought that only one exon 2 was present in the SMN gene(see, Lefebvre et al. Cell, 80:155-165 (1995)), the sequencing dataproved otherwise and the previously mentioned exon 2 in Lefebvre et alsupra is in fact composed of 2 exons separated by an additional intron,as illustrated in FIGS. 9 and 10. To avoid confusion in the renumberingof exons, the 2 exons in exon 2 are now referred to as exon 2a and exon2b.

All exon-intron bounderies displayed the consensus sequence found inother human genes and a polyadenylation consensus site is localized 550bp downstream from the stop codon (FIG. 10).

Starting from either YAC clones 121B8 or 595C11 (which contain theC-BCD541 and SMN genes respectively, (see, Lefebvre et al, supra) PCRamplification and sequence analysis of the introns showed threedifferences between SMN and C-BCD541 in addition to those previouslydescribed (by Lefebvre et al, supra). These included a base change inintron 6 (−45 bp/exon 7, atgt, telomeric; atat, centromeric) and twochanges in intron 7 (+100 bp/exon 7, ttaa, telomeric; ttag, centromericand at position +214 bp/exon 7, ttat, telomeric; ttgt, centromeric, FIG.10). The presence of both versions in a YAC clone containing both genes(YAC 920C9), and in the control population demonstrated that thesesubstitutions are locus-specific rather than due to polymorphism. Bandshifts on single strand conformtation polymorphism (SSCP) analysis ofthe PCR amplified intron 7 allowed SMN and its centromeric counterpart(C-BCD541) to be readily distinguished (see, FIG. 15).

In order to identify sequences potentially important for promoterfunction, the organization of the region surrounding exon 1 of the SMNand C-BCD541 genes was characterized. Based on restriction mapping,Southern blot hybridization and PCR amplification, exon 1 and the C272marker (D5F150S1, D5F150S2) were located in the same BgIII-EcoRIrestriction fragment of L-132 phage (FIG. 9). PCR amplification usingthe C272f primer and a reverse primer chosen in exon 1 was performed andthe amplified product was directly sequenced. Sequence analysis showedthat the (CA) repeat corresponding to the C272 marker are located 463 bpupstream from the putative ATG translation start site (FIG. 11).Comparative sequence analyses showed no discrepancy between the 5′ endsof the SMN gene and its centromeric counterpart (C-BCD541). In addition,sequence analysis showed the presence of putative binding sites for thefollowing transcription factors: AP-2, GH-CSE2, DTF-1, E4F1, HiNF-A,H4TF-1, β-IFN, Sp1 (FIG. 11; Faisst et al, Nucleic Acids Res., 20:3-26(1992)).

Besides isolating and characterizing the human SMN gene, the mousehomologue of the SMN gene was also cloned. Cross-species conservation ofhuman SMN gene with rodents has been shown in Lefebvre et al, supra andserved to isolate the mouse SMN gene. Screening of a mouse fetal cDNAlibrary using human SMN cDNA as a probe allowed the isolation of 2overlapping mouse cDNA clones, Sequence analysis of the clones revealedan 864 bp open-reading frame (ORF) (FIG. 12). The ORF encodes a putativeprotein of 288 amino acids (FIG. 12) with an homology of 83% with humanSMN amino acid sequence (FIG. 13).

Either the isolated human or the mouse SMN, the gene can be insertedinto various plasmids such as pUC18, pBr322, pUC100, λgHI, λ18-23, λZAP,λORF8, and the like. The methods for inserting genes into differentplasmid vectors are described by Sambrook et al supra. Variousmicroorganisms can be used to transform the vector to produce the SMNgene. For example, host microorganisms include, but are not limited to,yeast, CHO cells, E. coli, Bacillus subtilis and the like.

Once recombinantly produced, the human SMN protein or the mouse SMNprotein can be further purified from the host culture by methods knownin the art.

Besides recombinantly producing the SMN protein, the present inventionalso relates to the production of polyclonal and monoclonal antibodies.These methods are known in the art as evidenced by Sambrook et al supra.The monoclonal antibody can be obtained by the procedure of Kohler andMilstein, Nature, 256:495 (1975); Eur. J. Immunol., 6:511 (1976) orHarlow and Lane Antibodies, a Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, New York (1988), and can he used, forexample, in diagnosing SMA, as well as other motor neuron disorders.

Polyclonal rabbit antisera can also be generated against syntheticpeptides corresponding to any part of the SMN amino acid sequenceincluding the amino terminus and carboxy terminus. More specifically,the following peptides were synthesized based on the amino acid sequenceset forth in FIG. 1: N- G G V P E Q E D S V L F R R G T C- terminal S RS P G N K S D N I K P K terminal F R Q N Q K E G R C S H S L N

The synthetic peptide may be coupled to a carrier protein such asKeyhole limpet hemocyanin (KLH) through an amino- or carboxy-artificialcysteine residue that may be synthetically added to the desiredsequence. The cysteine residue is used as a linker to couple thesynthetic peptide to the carrier protein. The procedure utilized tocouple synthetic peptides to KLH is described by Green et al. Cell,28:477 (1982).

Approximately, 50-100 μg, preferably 100 μg of synthetic antigen isdissolved in buffer and emulsified with an equal volume of Freund'scomplete adjuvant. About 0.025 ml to 0.5 ml of emulsifiedantigen-adjuvant can be injected intramuscularly or intradermaly into arabbit. Four to six weeks later, the rabbit is boosted and 20-40 ml ofblood is drawn 7-10 days after each booster injection. The serum is thentested for the presence of antigen using RIA, ELISA orimmunoprecipitation. The positive antibody fractions may then bepurified, for example by absorption to protein A following the method ofGoudswaald et al, Scand. J. Immunol., 8:21 (1978).

More specifically, about 20 to 50 μg of antigen, prepared either by therecombinant techniques set forth above or synthetically made antigen isdiluted in about 100 μl of buffer and emulsified with an equal amount ofFreund's complete adjuvant. About 30-60, preferably 50 μl of theemulsified antigen-adjuvant is injected subcutaneously at four sitesinto mice. Four to six weeks later, the mice are boosted with anintraperitoneal injection of about 100 μl containing 5-10 μg of antigenSolubilized in buffer. The mice are bled from the mediam tail vein 7-10days after the boaster injection and the serum is tested for antibodyusing standard methods. Blood is then drawn every 3-4 days until theantibody titer drops.

Tissue, plasma, serum, cerebral spinal fluid and the like can be used todetect SMA disease using the above-described monoclonal or polyclonalantibodies via Western blot (1 or 2 dimensional) or ELISA. These methodsare known in the art as described by Sambrook et al, supra.

A method for detecting SMA as well as in ALS, ACM, and PLS patients whopossibly have these motor neuron disorders, is also encompassed by thepresent invention. This method involves extracting from a patientsuspected of having SMA. DNA from a sample. This sample may includesera, plasma, cerebral spinal fluid and the like. After extracting theDNA by known methods in the art, primers that are derived from exons 7and 8 of the SMN gene are used to amplify the DNA.

After amplification with the primer, the amplified product is subjectedto SSCP (Single Strand Conformation Polymorphism).

The gels are then subjected to autoradiography to determine if SMA ispresent in the sample.

More specifically, it has recently been discovered that in twelve casesof arthrogryposis multiplex congenita (AMC) associated with SMA, 6 outof 12 patients lacked the SMN gene.

A total of twelve unrelated patients including eight males and fourfemales of various geographic origins was selected for the study. Thepatients were chosen based on the criteria that these patients had:

(1) congenital joint contractures of at least two regions of the body(see, Stern, JAMA, 81:1507-1510 (1923));

(2) generalized muscle weakness with muscular atrophy and areflexiawithout extraocular involvement;

(3) electromyographic studies showed denervation and diminished motoraction potential amplitude; and

(4) muscle biopsies consistent with denervation with no evidence ofstorage material or other structural abnormalities (see, Munsat,Neuromuscular Disorders, 1:81 (1991)).

The study consisted of Dinucleotide Repeat Polymorphism Analysis and SMNgene analysis (see, Examples) based on DNA extracted from peripheralblood leukocytes, lymlphoblastoid cell lines or muscle tissue in alltwelve patients.

The data from this study is summarized in Table 1 below.

The diagnosis was made at birth with an uniform phenotype characterizedby a severe hypotonia, absence of movements except extraocular mobilityand contractures of at least two joints. The number of affected jointsand the severity of the postural defects varied from infant to infant,as set forth in Table 1. Decreased fetal movements were noted in 7 outof 12 (7/12) patients. Neonatal respiratory distress was observed in 9out of 12 (9/12) patients and facial involvement associated withmicrognathia was noted in 4 out of 12 (4/12) patients. Most of thecases, 8 out of 12 (8/12), died within the first month of life. Fourinfants are still alive. No family history was noted except in family 12in which both the child and her father were affected suggesting anautosomal dominant form of AMC.

Table 1 shows that the SMN gene was lacking on both mutant chromosomesin 6 out of 12 (6/12) patients (cases 1-6). Among them, 3 out of 6 (3/6)patients had a large inherited deletion involving both loci detected bymarkers C212 and C272 on one parental allele, the other parentalcarrying only one locus instead of the expected two, as shown in FIG.14.

Analysis of SMN exons did not reveal intragenic mutations in thepatients whose SMN gene showed no deletions (cases 7-12). Geneticanalysis showed that the disease gene in a family (case 9) was notlinked to chromosome 5q13 as both the affected and healthy siblingscarried the same 5q13 haplotype. These data strongly suggest that thepatients whose SMN gene showed no deletions were not linked to the 5q13SMA locus (cases 7-12).

Hitherto, arthrogryposis was regarded as an exclusion criterion in SMA(see, Munsat, supra). But the observation of SMN gene deletion in 6 outof 12 (6/12) patients (50%) strongly indicates that arthrogryposis ofneurogenic origin is related to SMA and that this subgroup and SMA areallelic disorders. Yet, AMC of neurogenic origin is a geneticallyheterogeneous condition since the disease gene was not linked to SMNlocus in 6 out of 12 (6/12) patients. Exclusion of chromosome 5q hasalso been shown in one family with two AMC-SMA patients, as described byLunt et al, J. Med. Genet., 29:273 (Abstract) (1992).

Thus, by dinucleotide Repeat Polymorphism Analysis and SMN geneanalysis, clinical diagnosis of AMC can be confirmed by the absence orinterruption of the SMN gene. The present invention now provides methodsto detect AMC either in live patients or in utero.

Yet another embodiment of the present invention is the detection of SMAusing specific oligonucleotide probes based on the nucleotide sequenceset forth in FIGS. 3, 10, or for the mouse SMA FIG. 12. If a patienttotally is lacking in the SMN gene, no hybridization to the specificprobe will occur. The hybridization conditions may vary depending uponthe type of sample utilized. It is preferable to conduct suchhybridization analysis under stringent conditions which are known in theart and defined in Sambrook et al supra. The oligonucleotide probes maybe labeled in any manner such as with enzymes, radioactivity and thelike. It is preferable to use radiolabeled probes.

In another embodiment of the present invention, the human SMN gene canbe utilized in conjunction with a viral or non-viral vector foradministration in vivo directly to the patients suffering from SMA orrelated motor neuron diseases or by administration in vitro in bonemarrow cells, epithelial cells fibroplasts, followed by administrationto the patient. See, for example Resenfeld et al, Science (1991) 252,pp. 431 to 434.

The present invention provides a method of detecting SMN gene defects orthe total lack of the SMN gene in a fetus. Amniotic fluid taken from thepregnant woman is subjected to SSCP analysis according to the methods ofthe present invention.

In order to further illustrate the present invention and advantagesthereof, the following specific examples are given, it being understoodthat the same are intended only as illustration and in nowiselimitative.

EXAMPLES Example 1

Construction of Phage Libraries from the 121B8, 595C11, and 903D1 YACClone.

Total yeast DNA from YAC clone 595C11 containing the telomeric locidetected by C212, C272 and C161, or YAC clone 121B8 containing thecentromeric loci detected by the same markers or 903D1 YAC clonecontaining both loci was purified and partially digested with Sau3A. DNAin the size range of 12 to 23 kb was excised after 0.5% Seaplaque GTGagarose gel electrophoresis and precipitated with ethanol afterβ-agarase digestion. After partial fill-in of the Sau3A site, DNA wassubcloned at the partially filled XhoI site of bacteriophage FIXIII(Stratagene). Clones of λ containing the microsatellite DNA markers C212(L-51), C272 (L-51, L-132), C171 (L-5, L-13). C161 (595B1), 11M1 (L-11).AFM157xd10(L-131) were digested either with EcoRI or HindIII or both andsubcloned into pUC18 plasmid vectors. Subclones from phages containingmarkers C212(p322), C272(132SE11), C161(He3), AFM157xd10(131xb4) andCMS1(p11M1) were used as probes.

Example 2

Pulsed Field Gel Electrophoresis Analysis

High molecular weight DNA was isolated in agarose plugs fromEpstein-Barr virus transformed lymphoblastoid cell lines establishedfrom controls and patients or from YAC clone as described. Plugs wererinsed twice for 30 min. each in 10-20 min vol. TE. The plugs wereequilibrated for 30′ at 4° C. with 0.3 ml of the appropriate restrictionenzyme buffer containing 0.1 mg/ml BSA (Pharmacia). Excess buffer wasthen removed and the plugs were incubated at the appropriate temperaturefor 16 h with 40 U restriction enzyme per reaction. DNA was digestedwith the restriction enzymes BssHII, EagI, SfiI, SacI, KpnI, SacII,SpeI. Separation of DNA fragments was performed using a CHEF-III-DR PFGEapparatus (Biorad). Fragments from 50 to 1200 kb were separated byelectrophoresis through 1% agarose Seakem, at 200 V for 24 h at 14° C.in 0.5×TBE running buffer using a 30′ to 70′ ramping pulse time. Theseparation of fragments from 5 to 100 kb was performed byelectrophoresis at 200 V for 19 h at 14° C. in 0.5×TBE buffer using a 5′to 20′ ramping pulse time. After treatment with 0.25N HCl for 20 min,pulsed field gels were blotted onto Hybond N+ Nylon membrane (Amersham)in 0.4N NaOH, 0.4M NaCl for 20 h. Probes were successively hybridized tothe same filters to ensure accurate data. Hybridizations were performedas described.

Example 3

YAC Library Screening

YAC libraries from CEPH were screened by PCR with microsatellites C212,C272, C171, CMS1, and C161. YAC genotypes were established byelectrophoresis of PCR products on denaturing polyacrylamide gels. YACsize was estimated by pulsed field gel electrophoresis.

Example 4

Southern Blot Analysis

DNA samples were extracted from either peripheral blood leukocytes orlymphoblastoid cell lines. DNA were digested with restriction enzymesEcoRI, HindIII, BgIII, XbaI, PvuII, XmnI, RsaI, PstI, BamHI, separatedby electrophoresis on an 0.8% agarose gel for Southern blotting andhybridized with radioactively labeled probes.

Example 5

Dinucleotide Repeat Polymorphisms

Genotypic data were obtained for the C212(D5F149S1, -S2), C272(D5F150S1,-S2) and C161(D5F153S1, -S2) dinucleotide repeat. Amplificationconditions were as follows: denaturation at 94° C., annealing at 55° C.,and extension at 72° C., 1 min each for 30 cycles. The procedure usedfor detection of dinucleotide repeat polymorphisms has been describedelsewhere.

Example 6

cDNA Clone and DNA Sequencing

Two million recombinants of a λgt10 human fetal brain library wereplated according to the manufacturer (Clontech). Prehybridization andhybridization was carried out in 10% Dextran Sulphate Sodium, 1 M NaCl,0.05 M Tris-HCl pH 7.5, 0.005 M EDTA and 1% SDS with 200 mg/ml shearedhuman placental DNA (Sigma) for 16 hours at 65° C. The filters werewashed in 0.1×SSEP−0.1% SDS at 65° C. and autoradiographs were performedfor 24 hours. The DNA of positive cDNA clones were purified, digestedwith EcoRI and subcloned in M13 bacteriophage. Single strand DNAs weresequenced using the DyeDeoxy™ Terminator Cycle Sequencing Kit protocolsupplied by Applied Biosystems, Inc. and analyzed on a ABI model 373ADNA automated sequencer. To obtain the 3′ end of the cDNA along with itspoly(A)⁺ tail, PCR-amplification of a lymphoblastoid cell line cDNAlibrary was performed using specific primer complementary to the 3′ endof the clones and primer specific to the vectors arms of the cDNAlibrary as previously described (Fournier B., Saudubray J. M., BenichouB. et al, 1994, J. Clin. Invest. 94:526-531). Specific PCR-products weredirectly sequenced with both primers using the DyeDeoxy™ TerminatorCycle Sequencing Kit protocol supplied by Applied Biosystems, Inc. andanalyzed on a ABI model 373A DNA automated sequencer.

Example 7

Isolation of RNA and Northern Blot Analysis

mRNA from lymphoblast cell lines of controls and SMA patients wereisolated with the QuickPrep mRNA purification kit (Pharmacia) accordingto the supplier's procedure. Total RNA was prepared following thesingle-step RNA isolation method described by Chomczynski and Sacchi(Analytic Biochemistry, 162:156-159 (1987)). The total RNA preparationwas treated with RQ1-DNAse (Promega) to remove any contaminating genomicDNA. Northern blots were made from mRNA and total RNA by electrophoresisthrough 1.5% seakem agarose gel containing methyl mercuric hydroxide andtransferred to positively charged membrane in 20×SSC and heated for 2hours at 80° C. ³²P-radiolabeled DNA probes were synthesized by a randompriming method according to the manufacturer (Boehringer), andhybridized in a solution containing 5×SSEP, 1% SDS, 5× Denhardt's for 16hours at 65° C. The membranes were washed to a final stringency of0.1×SSEP, 0.1% SDS at 65° C. for 10 min. Autoradiography was at −80° C.with intensifying screens and Kodak XAR films for 2 to 10 days. Theamount of mRNA was normalized with a b-actine cDNA probe. Theautoradiographs were scanned at 600 nm in computerized densitometer(Hoeffer Scientific Instruments, San Francisco). A Northern blot withpolyA+ RNA from several huma tissues was purchased from Clontech.

Example 8

Reverse Transcriptase-Based PCR Amplification and Sequencing

Each PCR amplification was carried out in a final volume of 20 ml onsingle-strand cDNAs synthesized from the random hexamers-primed reversetranscription (Promega). The PCR reactions included 2 picomoles offorward and reverse primers and 1 unit Taq polymerase in the reactionbuffer recommended by Perkin Elmer/Cetus. Parameters for PCRamplification consisted in 1 min at 94° C., 1 min at 55° C. and 1 min at72° C. for 30 cycles followed by a final extension period of 10 min at72° C. Parameters for PCR amplification consisted in 1 min at 94° C., 1min at 55° C. and 1 min at 72° C. for 30 cycles followed by a finalextension period of 10 min at 72° C. The PCR products were cut fromacrylamide gel and eluted in 100 ml of TE buffer. The diluted fragmentswere reamplified with the same primers prior direct sequencing. The PCRamplification products were cut from acrylamide gel and eluted in 100 mlof TE buffer. The diluted fragments were reamplified prior to directsequencing with both primers using the DyeDeoxy™ Terminator CycleSequencing Kit protocol supplied by Applied Biosystems, Inc. andanalyzed on a ABI model 373A DNA automated sequencer. Six sets ofprimers along the cDNA sequence were used to amplify DNA products forsequence analysis.

Example 9

Computer-Assisted Analysis

Sequence homology analysis with both nucleotide and protein sequencesfrom 541C were performed using FASTA and BLAST through the CITI2 Frenchnetwork (Dessen P., Fondrat C., Velencien C., Mugnoer C., 1990, CABIOS;6:355-356).

Example 10

SSCP Analysis

For single strand conformation polymorphism (SSCP) analysis, DNA fromperipheral leukocytes (200 ng) was submitted to PCR amplification usingunlabelled primers (20 μM) in 25 μl amplification mixture containing 200μM dNTPs, 1 unit of Taq polymerase (Gibco-BRL) and 0.1 μl of a ³²P dCTP(10 mCi/ml, NEN). Amplified DNA was mixed with an equal volume offormamide loaded dye (95% formamide, 20 mM EDTA, 0.05% bromophenol blue,0.05% xylene cyanol). The samples (5 μl) were denatured for 10 mn at 95°C. and loaded onto a polyacrylamide gel (Hydroling MED, Bioprobe) andelectrophoresed at 4° C. for 18 to 24 hours at 4 W. Gels weretransferred onto 3 MM Whatman paper, dried and autoradiographed withKodak X-OMAT films for 24 hours. To amplify the DNA sequence containingthe divergence of exon 7 oligonucleotides R111 (5′AGACTATCAACTTAATTTCTGATCA 3′) and 541C770 (5′°°TAAGGAATGTGAGCACCTTCCTTC3′)—were used. To amplify the DNA sequence containing the divergence ofexon 8 oligonucleotides 541C960 (5′ GTAATAACCAAATGCAATGTGAA 3′) and541C1120 (5′ CTACAACACCCTTCTCACAG 3′) were used.

Example 11

Cloning of the Human SMN Gene

Total yeast DNA from YAC clone 595C11 was purified via the method ofSambrook et al supra and partially digested with restriction enzymeSau3A. DNA in the 12-23 kD size range was excised after 0.5% sea plagueGTG agarose gel electrophoresis and precipitated with ethanol afterβ-agarase digestion. After partial fill-in of the Sau3A site, DNA wassubcloned at the partially filled XhoI site of bacteriophage FIXII(Stratagene).

The full-length BCD541 cDNA was used as a probe to screen the FIXIIphage library under conditions set forth in Sambrook et al, supra.

These phages, named M-132, L-5 and L-13 spanned the entire SMN gene asconfirmed by restriction mapping using HindIII, EcoRI and BgIII (see,FIG. 9) and Southern blot analysis.

The phages were then sequenced as described in Example 8. Once the genewas sequenced, it was then cloned into a pUC18 vector and recombinantlyreproduced in large quantities that were purified for further use.

Example 12

Cloning of the Mouse SMN Gene

A mouse fetal cDNA library was screened using the coding sequence of thehuman SMN cDNA as a probe according to Sambrook et al, supra.

Two overlapping mouse cDNA clones were found that had the entiresequence of mouse SMN, as revealed by sequencing methods described inExample 8 after being cloned into a pUC18 vector and M13 vectors.

Example 13

Transgenic Mouse

Transgenic mice containing multiple normal SMN genes OR SMN geneslacking exon 7 are produced by the methods according to Lee et al,Neuron, 13:978-988 (1994). The transgenic animals are then tested andselected for the overexpression of the SMN gene or SMN gene lacking exon7 via Southern, and/or Northern blots using the probes described in thepresent invention or by screening with antibodies described in thepresent invention in a Western blot.

Transgenic mice containing abnormal SMN genes are obtained by homologousrecombination methods using mutated SMN genes as described by Kühn etal, Science, 269:1427-1429 (1995) and Bradley, Current Opinion inBiotechnology, 2:823-829 (1991). The transgenic animals are then testedand selected for the overexpression of the SMN gene via Southern, and/orNorthern blots using the probes described in the present invention or byscreening with antibodies described in the present invention in aWestern blot selected for the abnormal SMN gene.

Example 14

Polyclonal Antibodies

100 μg of a synthetic antigen having sequence:

-   -   N-terminal G G V P E Q E D S V L F R R G T C-terminal        was dissolved in buffer and emulsified with an equal volume of        Freund's complete adjuvant. 0.5 ml of the emulsified synthetic        antigen-adjuvant was injected intramuscularly into a rabbit.        Five weeks later, the rabbit was boosted and 20-40 ml of blood        was drawn 8 days after each booster injection. The serum was        then tested for the presence of antigen using RIA.

Polyclonal antibodies were also prepared by the same methods using thefollowing sunthetic antigens: N- S R S P G N K S D N I K P K C- terminalF R Q N Q K E G R C S H S L N terminal

Example 15

Gene Therapy

Using the adenovirus construct described by Ragot et al, Nature. Vol.361 (1993), the normal SMN gene was inserted therein and injectedintramuscularly into a patient lacking this gene. The patient ismonitored using SSCP analysis as described in Example 10 above.

While the invention has been described in terms of various preferredembodiments, the skilled artisan will appreciate that variousmodifications, substitutions, omissions and changes may be made withoutdeparting from the spine thereof. Accordingly, it is intended that thescope of the present invention be limited solely by the scope of thefollowing claims, including equivalents thereof. TABLE I Case 1 2 3 4 56 7 8 9 10 11 12* Sex m f m m m m m m m f f f Age of death d 8 d 6 d 1 d25 d 11 d 13 4 m >3 y >3 y d 20 >9 y >16 m Fetal movements + + − + − − +− + − + + diminished Hypotonia + + + + + + + + + + + +Respiratory + + + + + + + − + + − − Involvement Neurogenic (EMG)? + + + + + nd + + + + + Muscle Atrophy (MB) + + + + + + + + + + + +Contractures Hips − − − − − + − + − − + + Knees + + + + + + − + − − + +Ankles + − − + − − − + + + + + Elbows − + + + − − + + − − − − Wrists −− + − + + + + − + − − Fingers − + − − + + − − − − − − Associated Signsfacial Ao. Co − − − − fract. − facial facial facial − micro. micro.micro. micro. C212/C272 markers + + del del + del + + unlink + + + SMNgene del del del del del del + + + + + +Abbreviations: +, present; −, absent; Ao. Co, aortic coartation; Fract.,bone fracture, Facial. microg, facial involvement with micrognathia; nd,not done.*Both the child and her father were affected.

1. An isolated human survival motor neuron (SMN) protein.